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The U.S. Army and U. S. Army Public Health Center are dedicated to protecting the health, and readiness of Department of the Army Service Members, civilians, and contractors. Despite implementation of health programs, policies and tobacco control interventions, the advent of electronic nicotine delivery systems (ENDS), including electronic cigarettes (e-cigs), represent unregulated and poorly defined systems to supplant or substitute use of conventional nicotine products (e.g., cigarettes and pipe tobacco). E-cigs present unique challenges to healthcare officials vested in preventive medicine. The health impact of an e-cig and vaping on an individual's acute or chronic disease susceptibility, performance and wellness, is fraught with uncertainty. Given the relatively recent emergence of e-cigs, high-quality epidemiological studies, and applied biological research studies are severely lacking. In sparsely available epidemiological studies of short-term cardiovascular and respiratory health outcomes, any attempt at addressing the etiology of acute and chronic health conditions from e-cig use faces incredible challenges. Until relatively recently, this was complicated by an absent national regulatory framework and health agency guidance on the manufacture, distribution, selling and use of e-cigs or similar ENDS devices and their chemical constituents. Two key issues underpin public health concern from e-cig use: 1) continued or emergent nicotine addiction and potential use of these devices for vaping controlled substances; and 2) inadvertent sudden-onset or chronic health effects from inhalational exposure to low levels of complex chemical toxicants from e-cig use and vaping the liquid. Herein, the health impacts from e-cig vaping and research supporting such effects are discussed.

Fischer-Tropsch (FT) Synthetic Paraffinic Kerosene (SPK) jet fuel is a synthetic organic mixture intended to augment petroleum-derived JP-8 jet fuel use by the U.S. armed forces. The FT SPK testing program goal was to develop a comparative toxicity database with petroleum-derived jet fuels that may be used to calculate an occupational exposure limit (OEL). Toxicity investigations included the dermal irritation test (FT vs. JP-8 vs. 50:50 blend), 2 in vitro genotoxicity tests, acute inhalation study, short-term (2-week) inhalation range finder study with measurement of bone marrow micronuclei, 90-day inhalation toxicity, and sensory irritation assay. Dermal irritation was slight to moderate. All genotoxicity studies were negative. An acute inhalation study with F344 rats exposed at 2000 mg/m

The subchronic toxicity of sodium tungstate dihydrate aqueous solution in male and female Sprague-Dawley rats was evaluated by daily oral gavage of 0, 10, 75, 125, or 200 mg/kg/d for 90 days. Measured parameters included food consumption, body weight measurements, hematology, clinical chemistry, and histopathological changes. There was a significant decrease in food consumption and body weight gain in males at 200 mg/kg/d from days 77 to 90; however, there was no effect in food consumption and body weights in females. There were no changes in the hematological and clinical parameters studied. Histopathological changes were seen in kidney of male and female and epididymis of male rats. Histopathological changes were observed in the kidneys of male and female rats dosed at 125 or 200 mg/k/d consisting of mild to severe cortical tubule basophilia in 2 high-dose groups. Histological changes in epididymides included intraluminal hypospermia with cell debris in the 200 mg/kg/d dosed male rats. Histopathological changes were observed in the glandular stomach including inflammation and metaplasia in the high-dose groups (125 or 200 mg/kg/d) of both sexes of rats. Based on histopathology effects seen in the kidneys, the lowest observable adverse effect level was 125 mg/kg/d and the no observable adverse effect level was 75 mg/kg/d in both sexes of rats for oral subchronic toxicity.

Thiodiglycol (TDG) is the main product of sulfur mustard hydrolysis and is an environmental contaminant. Subacute and subchronic oral toxicity studies with TDG were conducted in Sprague-Dawley rats. Neat TDG was administered by gavage at doses of 157, 313, 625, 1250, 2500, 5000, and 9999 mg/kg/d, 5 days per week, for 14 days. In the 14-day study, decreased body weight and food consumption were observed at 5000 mg/kg/d. In the 90-day study, rats received neat TDG at doses of 50, 500, or 5000 mg/kg/d for 5 days per week. A fourth group served as a sham control. Individual body weight and food consumption were measured weekly. At termination of the experiment, urine, blood, and tissue samples were collected. Rats displayed significant decreased body weight with no effect on food consumption following administration of TDG at 5000 mg/kg/d. Both male and female rats showed significant increased kidney weights at 5000 mg/kg/d. The organ to body weight ratios increased significantly for liver, kidneys, testes, and brain in males and adrenals in females for 5000 mg/kg/d. At all doses of TDG, hematological and clinical parameters and tissue histopathology remained unaltered. The no observed adverse effect level (NOAEL) for oral subchronic toxicity was 500 mg/kg/d. Benchmark dose (BMD) was derived from the decreased gain in body weight that was seen in male rats. A BMD based on a 10% decrease in body weight was 1704 mg/kg/d, and the lower confidence limit on the dose BMD, the BMDL, was 372 mg/kg/d.

The consideration of multiple or cumulative sources of exposure to a chemical is important for adequately protecting human health. This assessment demonstrates one way to consider multiple or cumulative sources through the development of a relative source contribution (RSC) factor for the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), using the Exposure Decision Tree approach (subtraction method) recommended by the U.S. Environmental Protection Agency. The RSC factor is used to ensure that the concentration of a chemical allowed by a regulatory criterion or multiple criteria, when combined with other identified sources of exposure common to the population of concern, will not result in unacceptable exposures. An exposure model was used to identify relevant potential sources for receptors. Potential exposure pathways include ingestion of soil, water, contaminated local crops and fish, and dermal contact with soil and water. These pathways are applicable only to areas that are in close pr...

Ethylenediamine dinitrate (EDDN) and diethylenetriamine trinitrate (DETN) are relatively insensitive explosive compounds that are being explored as safe alternatives to other more sensitive compounds. When used in combination with other high explosives they are an improvement and may provide additional safety during storage and use. The genetic toxicity of these compounds was evaluated to predict the potential adverse human health effects from exposure by using a standard genetic toxicity test battery which included: a gene mutation test in bacteria (Ames), an in vitro Chinese Hamster Ovary (CHO) cell chromosome aberration test and an in vivo mouse micronucleus test. The results of the Ames test showed that EDDN increased the mean number of revertants per plate with strain TA100, without activation, at 5000μg/plate compared to the solvent control, which indicated a positive result. No positive results were observed with the other tester strains with or without activation in Salmonella typhimurium strains TA98, TA1535, TA1537, and Escherichia coli strain WP2 uvrA. DETN was negative for all Salmonella tester strains and E. coli up to 5000μg/plate both with and without metabolic activation. The CHO cell chromosome aberration assay was performed using EDDN and DETN at concentrations up to 5000μg/mL. The results indicate that these compounds did not induce structural chromosomal aberrations at all tested concentrations in CHO cells, with or without metabolic activation. EDDN and DETN, when tested in vivo in the CD-1 mouse at doses up to 2000mg/kg, did not induce any significant increase in the number of micronuclei in bone marrow erythrocytes. These studies demonstrate that EDDN is mutagenic in one strain of Salmonella (TA100) but was negative in other strains, for in vitro induction of chromosomal aberrations in CHO cells, and for micronuclei in the in vivo mouse micronucleus assay. DETN was not genotoxic in all in vitro and in vivo tests. These results show the in vitro and in vivo genotoxicity potential of these chemicals.

Recognition of the occupational hazards from exposure to the propellants hydrazine and monomethylhydrazine (MMH) has led to research into less toxic alternatives. Two hypergolic compounds, dimethylamino-2-ethylazide (DMAZ) and N,N,N′,N′-tetramethylethanediamine (TMEDA), have been identified as possible replacements for MMH. We have obtained genotoxicity data for these compounds from in vitro and in vivo studies. DMAZ did not produce any mutagenic effects at concentrations up to 5 mg/plate in the TA98 and TA1537 strains of Salmonella typhimurium and in an Escherichia coli (WP2 uvrA ) strain, with or without metabolic activation, but did produce a positive response in the TA100 and TA1535 strains, both with and without metabolic activation. TMEDA was found not to be mutagenic in any of the bacterial strains tested ( Salmonella TA98, TA100, TA1535, TA1537 and E. coli, WP2 uvrA ), with or without metabolic activation. DMAZ did not induce structural chromosomal aberrations at levels up to 5 mg/mL in Chinese hamster ovary (CHO) cells, with or without metabolic activation. TMEDA produced a positive response in this system, with or without metabolic activation, but only at the highest concentration, 5 mg/mL. However, according to the OECD guideline TG 473, the compound is considered to be negative in the CHO chromosomal aberration assay, since the compound was not clastogenic at 0.01 M (1.140 mg/mL). DMAZ and TMEDA, when tested in vivo in the CD-1 mouse at doses up to 500 and 250 mg/kg, respectively, did not induce micronuclei in bone marrow erythrocytes. These studies demonstrate that DMAZ is mutagenic in specific strains of Salmonella . However, both compounds were negative for induction of chromosomal aberrations in CHO cells in vitro and in the in vivo mouse micronucleus assay.

Cyclotrimethylenetrinitramine (RDX) has been used extensively as an explosive in military munitions. Mechanisms for seizure production, seen in past animal studies, have not been described. Increased calcium levels contribute to excitotoxicity, so in this study neuroblastoma cells are loaded with calcium-indicating dye before application of 1.5 µM to 7.5 mM RDX, with fluorescence recorded for 30 cycles of 11 seconds each. The lowest concentration of RDX increases calcium fluorescence significantly above baseline for cycles 2 to 8; millimolar concentrations increase calcium fluorescence significantly above baseline for cycles 2 to 30. Increases in calcium, like those of 200 nM carbachol, are prevented with 10 mM of calcium chelator ethylene glycol-bis(β-aminoethyl ether)-N,N,N,N tetra-acetic acid (EGTA, tetrasodium salt). Calcium channel blocker verapamil (20 μM), Ca2+-ATPase inhibitor thapsigargin (5 μM), and general membrane stabilizer lidocaine (10 mM) partially attenuate carbachol- and RDX-induced increases in calcium, suggesting that RDX transiently increases intracellular calcium by multiple mechanisms.

Water plays a key role in enhancing the permeability of human skin to many substances. To further understand its ability to potentially increase the bioavailability of soil contaminants, artificial sweat was applied to excised pig skin prior to dosing with munition-contaminated soils. Skin was mounted in chambers to allow simultaneous measurement of evaporation and penetration and to control air flow, which changed the dwell time of skin surface water within a l-h period post application of test materials. Additional variables included type of compound, aging of spiked soil samples, and carbon content of soil. To this end, the evaporation and skin penetration of C-14 labeled hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX), 2,6-dinitrotoluene (26DNT), and 2,4,6-trinitrotoluene (TNT) were determined from two soil types, Yolo, having 1.2% carbon, and Tinker, having 9.5% carbon. RDX soil samples aged 27 mo and 62 mo were compared to freshly spiked soils samples. Similarly, 26DNT samples aged 35-36 mo and TNT samples aged 18 mo were compared to freshly spiked samples. Approximately 10 microg/cm(2) of radiolabeled compound was applied in 10 mg/cm(2) of soil. Radiolabel recovered from the dermis and tissue culture media (receptor fluid) was summed to determine percent absorption from the soils. Radiolabel recovered from vapor traps determined evaporation. Mean skin absorption of all compounds was higher for low-carbon soil, regardless of soil age and skin surface water as affected by air flow conditions. For 26DNT, a simultaneous increase in evaporation and penetration with conditions that favored enhanced soil hydration of freshly prepared samples was consistent with a mechanism that involved water displacement of 26DNT from its binding sites. A mean penetration of 17.5 +/- 3.6% was observed for 26DNT in low-carbon soil, which approached the value previously reported for acetone vehicle (24 +/- 6%). 26DNT penetration was reduced to 0.35% under dryer conditions and to 0.08% when no sweat was applied. When soil hydration conditions were not varied from cell to cell, air flow that favored a longer water dwell time increased penetration, but not evaporation, consistent with a mechanism of enhanced skin permeability from a higher hydration state of the stratum corneum. Profiles of 26DNT penetration versus air flow conditions were exponential for freshly prepared soil samples, suggesting strong and weak binding sites; corresponding profiles of 26DNT penetration from aged samples were linear, suggesting a conversion of weak to strong binding sites. Absorption and evaporation was less than 5% for TNT and less than 1% for RDX, regardless of soil type and age. Fresh preparations of RDX in Tinker soil and aged samples of TNT in Yolo soil showed a significant decrease in skin absorption with loss of surface moisture. The penetration rate of radiolabel into the receptor fluid was highest during the 1-2 h interval after dosing with 26DNT or TNT. High-performance liquid chromatography (HPLC) analysis of 26DNT in receptor fluid at maximum flux indicated no metabolism or breakdown. For TNT, however, extensive conversion to monoamino derivatives and other metabolites was observed. Relatively little radioactivity was found in the dermis after 26DNT and TNT applications, and dermal extracts were therefore not analyzed by HPLC. RDX was not sufficiently absorbed from soils to allow HPLC analysis. This study has practical significance, as the use of water for dust control at remediation sites may have the unintended effect of increasing volatilization and subsequent absorption of soil contaminants. Soil in contact with sweaty skin may give the same result. Skin absorption of 26DNT from soil was over 50-fold higher than the value for dryer skin and over 200-fold higher than the value obtained when there was no sweat application. While the hydration effect was less dramatic for RDX and TNT, soil contaminants more closely matching the physical properties of 26DNT may be similarly affected by hydration.

The study reported herein examined the metabolism of 14C-labeled hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) resulting from a single oral gavage of 5 ml/kg to male and female Yucatan miniature pigs (43 mg/kg, 56 microCi/kg in 0.5% carboxymethylcellulose in water). Blood, urine, and feces were collected at selected times of 1, 6, 12, and 24 h postdose. At 24 h postdose, liver samples were collected. Blood, plasma, liver, and excreta were analyzed for total RDX-derived radioactivity and metabolites were identified. Urine was the major route of elimination of 14C-RDX-derived radioactivity in both males and females. Relatively low levels of radioactivity were found in gastrointestinal contents and in feces, suggesting nearly complete absorption of 14C-RDX following an oral dose. Analysis of urine by liquid chromatography-mass spectrometry (LC/MS) identified quantifiable levels of two ring-cleavage metabolites, 4-nitro-2,4-diazabutanal and 4-nitro-2,4-diaza-butanamide, as well as parent RDX. The 4-nitro-2,4-diazabutanal, was seen in earlier studies of aerobic metabolism of RDX. The 4-nitro-2,4-diaza-butanamide, an amide, was not previously reported but was tentatively identified in this study. Analysis by a more sensitive method (LC/MS/MS) also showed trace amounts of the RDX metabolites 1-nitroso-3,5-dinitro-1,3,5-triazacyclohexane (MNX) (in both male and female urine) and 1-nitro-3,5-dinitroso-1,3,5-triazacyclohexane (DNX) (in male urine). Analysis of plasma by LC/MS/MS also revealed quantifiable levels of RDX and trace levels of MNX, DNX, and 1,3,5-trinitroso-1,3,5-triazacyclohexane (TNX). None of the liver extracts showed quantifiable levels of RDX or any identifiable metabolites. Most of the radioactivity was in the form of water-soluble high-molecular-weight compounds. RDX when given orally to pigs was rapidly metabolized by loss of two nitro groups followed by ring cleavage.

Thiodiglycol (TDG) is used commercially as a solvent in antifreeze solutions, dyestuffs for printing, and as part of the process by which polyvinyl chloride is manufactured. It is also either a starting material or a precursor in the production of the chemical warfare agent sulfur mustard. TDG toxicity data for birds, amphibians, and reptiles were lacking. For mammals, acute, subacute, and subchronic studies were available representing two species in two orders. Adverse effects of TDG include reduced body weight, as well as increased kidney weight, increased urine output, and decreased urine pH, which may indicate functional deficits in the kidney. Reproductive and developmental effects were observed in one species representing one order and include decreased maternal and fetal body weight and decreased maternal food consumption. No and low observed adverse effect levels (NOAEL and LOAEL) of TDG for mammals of 129 mg/kg/day and 387 mg/kg/day respectively, were developed. The sole inhalation study suggests exposures of 5.4 mg/L TDG or less are not likely toxic. Dermal studies gave sufficient data for TRV development resulting in a NOAEL of 118 mg/kg and a LOAEL of 1,180 mg/kg.

1,3,5-Trinitrobenzene (TNB) is a nitroaromatic compound that is used as an explosive and is released to the environment during the manufacturing process and as a degradation or by-product of 2,4,6-trinitrotoluene (TNT) manufacture. TNB detected in soils and water can be hazardous to animals. The reported acute lethal dose (LD) LD50 values were in the range of 450 to 730 in rats, mice, and guinea pigs. TNT promotes hematological, testicular, and neurological effects in rats and white-footed mice in acute, subchronic, and chronic toxicity studies. The oral dietary no observed adverse effect level (NOAEL) for rats was 2.68 milligrams per kilogram (mg/kg), and the lowest observed adverse effect level (LOAEL) was 13.31 mg/kg in chronic toxicity studies. These values are considered the Toxicity Reference Values (TRVs) for mammals following ingestion since these values were below the NOAEL for reproductive and development effects. Uncertainty factors were not used. The derivation of benchmark dose (BMD) curves failed to fit data points to derive the benchmark dose lower confidence limit (BMDL). The toxicity data on wild mammals, amphibians, avian, and reptile species were unavailable to develop TRVs.

Mustard gas, or HD, refers to two classes of blister-causing chemical agents, sulfur mustard and nitrogen mustard, that are highly toxic, alkylating chemical agents that produce blisters on exposed skin and cause extreme pain and suffering to their victims. It is toxic when exposed by oral, inhalation, and dermal route. HD also has mutagenic and carcinogenic properties. The subchronic oral no observed adverse effect level (NOAEL) of 0.07 mg/kg and lowest observed adverse effect level (LOAEL) of 0.2 milligrams per kilogram (mg/kg) were reported in rats. The LOAEL of 0.02 mg/kg was reported for reproductive effects in rats. The accidental spillages are the potential point source of exposure for wildlife. Spilled mustard gas could persist for weeks in the environment because it is heavier than air, with moderate volatility. Wildlife exposure is expected to be primarily via contact with airborne gas. No Toxicity Reference Values (TRV) could be derived for mammalian, avian, amphibian, and reptilian species due to insufficient data.

Trinitrotoluene (2,4,6-trintrotoluene, TNT) is an extensively used explosive, which has been detected in soil and water at military sites and at munitions manufacturing and training operations. TNT contamination may be hazardous to animal health. The toxicity data of TNT on wildlife were evaluated using the benchmark dose procedure on sensitive adverse effects described for mammals and birds. Adjustments to the no observed adverse effect (NOAEL) and lowest observed adverse effect (LOAEL) values were used to derive toxicity reference values (TRVs) for amphibians and reptiles. The TRVlow and TRVhigh of 0.2 and 0.3 mg/kg/day, respectively, for mammals were derived using body mass changes in dogs as a result of oral TNT exposure. The TRVlow and TRVhigh of 60 and 90 mg/kg/day were calculated for birds based upon changes in plasma hemoglobin concentration related to dose. The TRVhigh and TRVlow of 40 and 4 mg/kg soil were determined for amphibians using the LOAEL and NOAEL values reported for microcosm exposures conducted in red-backed salamanders with a low level of confidence. Plasma hemoglobin content was modeled in Western Fence Lizards where TRVhigh and TRVlow for reptiles were derived using benchmark dose of 19 and 14 mg/kg/day, respectively, with a low level of confidence. Together, these values provide the tools to enable risk assessors to screen for adverse effects of TNT in terrestrial environments.

This chapter presents the wildlife toxicity assessment for nitroguanidine (NQ), a stable explosive compound used as a propellant in cartridges. This wildlife toxicity assessment addresses only toxicity to mammals, as data for amphibians, reptiles, and birds were lacking. Acute, chronic, and reproductive studies represented three mammalian species (rats, mice, and rabbits) in two orders, and showed limited toxicity of NQ to mammals. Water consumption was the most consistent effect. At high doses (1,000 mg/kg/day) other affects included death, reduced body weight gain, and reduced food consumption, which were attributed to decreased palatability and interference with digestion at 1,000 mg/kg/day. In developmental/reproduction studies, reduced fetal body weight and increased incidence of minor developmental alterations were noted. Thus, in mammals, the no and low observed adverse effect levels (NOAEL and LOAEL) for NQ were estimated to be 316 mg/kg/day and 1,000 mg/kg/day, respectively.

The synthetic nitro-aromatic compound m-nitrobenzene (m-DNB) is used in explosives, dyes, and as an intermediate in the synthesis of chemicals. It is found in the environment as a byproduct of trinitrotoluene (TNT). Environmental contamination by m-DNB may be hazardous to animals and is acutely toxic since it produces hematological, testicular, and neurological effects in animals. The toxicity data of DNB was evaluated for the derivation of wildlife toxicity reference values (TRV) for m-DNB that are protective to wildlife. The toxicity data on wildlife is limited to only mammalian species. The TRV for mammals is derived from the testicular and reproductive toxicity data of rats, which are sensitive at a no observed adverse effect level (NOAEL) of 0.75 milligrams per kilogram per day (mg/kg/day) for significant reproductive performance that were used to develop a NOAEL of 0.04 mg/kg/day and a lowest observed adverse effect level (LOAEL) of 0.2 mg/kg/day.

Benzo[a]pyrene (B[a]P) is one of a large number of polycyclic aromatic hydrocarbons (PAHs) that are formed during the incomplete combustion of organic matter. It is the most well-studied PAH, a cancer-inducing substance for which toxicological data serve as quantitative benchmarks for the entire carcinogenic subgroup. In general, Toxicity Reference Values (TRVs) are based on noncancer effects, of which there is paucity of studies for B[a]P. The TRVs presented in this chapter are intended to serve as benchmarks for screening-level ecological risk assessments. The reproductive toxicity data was used to develop TRV for mammals. The NOAEL of 1 mg/kg/day and LOAEL of 10 mg/kg/day were developed for class Mammalia of ingestion TRV with medium confidence. The experimental data on the toxicity of B[a]P is not relevant to the development of wildlife TRVs for the compound, for avian, reptilian, or amphibian species.

Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (or high melting explosive, HMX) is an N-nitrated, eight-member heterocyclic ring compound that is used in military munitions. Occupational and environmental contamination might be due to the manufacturing process; environmental discharge of waste waters; during the manufacture of explosives; and in load, assembly, and pack (LAP) activities at U.S. Army ammunition plants and other military installations. The acute oral LD50 values range from 2 to 6 mg/kg in rats and mice. The acute NOAEL-based approximate TRV of 0.5 mg/kg and LOAEL-based TRV of 1 mg/kg were determined. The NOAEL for ingestion TRV for mammalian omnivorous was 62.5 mg/kg/d and the NOAEL of 9 mg/kg/d was derived from a subchronic study of mice. The amphibian ingested NOAEL-based TRV was 197 mg/kg. The limited avian and reptilian toxicity data warrants to develop TRVs for the wildlife species.

Lewisite is a chemical warfare agent that was used during World War I, has been stockpiled in the United States, and has been used by other nations. Its unplanned release into the environment is possible. It degrades rapidly to lewisite oxide. Its potent vesicant produces rapid toxic effects from acute oral, inhalation, and dermal exposure routes. The oral subacute rabbit study of an estimated 7-day dose of no observed adverse effect level (NOAEL) of 0.07 milligrams per kilogram per day (mg/kg/day) and a lowest observed adverse effect level (LOAEL) of 0.2 mg/kg/day enabled derivation of Toxicity Reference Values (TRVs) of 0.004 mg/kg/day and 0.03 mg/kg/day, respectively. The inhalation TRV was derived from a vapor exposure lethal concentration (LCt 50 ) of 500 mg/min/m 3 for NOAEL of 5.0 mg/min/m 3 and LOAEL of 0.25 mg/min/m 3 . The dermal TRVs that were derived on the LD 50 of 15 mg/kg were 0.15 mg/kg of NOAEL and 0.75 mg/kg of LOAEL. No data is available on avian wildlife, amphibians, and reptiles.

1,3,5-Trinitrobenzene (TNB) is a munitions chemical that causes gliovascular lesions in the brain stem of rats similar to those produced by thiamine deficiency and nitroaromatic compounds, including m-dinitrobenzene. To identify neuropathic indices of toxicity, the effects of varying concentrations (0 to 2 mM) of TNB on cytotoxicity and cellular metabolic activity were examined using cultured astrocytes from Fischer-344 rats. The cytotoxicity was assessed by lactate dehydrogenase (LDH) leakage into the culture medium. Astrocyte metabolic activity was assessed by measuring the conversion of a tetrazolium salt to a formazan product. Additionally, the effects of oxidative stress on cellular metabolic activity - were determined by varying oxygen tension via alteration of culture media depth. In vitro, the toxic concentration 50% (TC50) of TNB, which induced cell death, was 16 μM following a 24-h exposure. The concentration of TNB that reduced cellular metabolic activity by 50% was 29 μM following a 24-h exposure. Varying the depth of the culture media did not influence the cellular metabolic activity in control or TNB-treated astrocytes. These results support the hypothesis that TNB induced neurotoxicity could partially be mediated via injury to astrocytes, a major component of the blood-brain barrier.

The chronic toxicity of 1,3,5-trinitrobenzene (TNB) in male and female Fischer 344 (F344) rats was evaluated by feeding a diet containing 0, 5, 60, and 300 ppm of TNB for 2 years. The calculated average TNB intake over 2 years for males and females was 0.22, 2.64,13.44 and 0.23, 2.68, 13.31 mg/kg body weight (BW)/day respectively. Terminal body weights were decreased and water intake was increased in both sexes (300 ppm), whereas food consumption was decreased in males (60 and 300 ppm groups) only. The relative spleen weights were significantly decreased in both sexes (300 ppm), whereas the relative brain weights were increased in females only (300 ppm). Hematological effects were not observed in animals killed at the 2-year time point, except significant decrease in the mean corpuscular hemoglobin (MCH) in males (300 ppm) and in females (60 and 300 ppm). Methemoglobin levels were increased in both sexes in the high dose group. Histopathological examination showed treatment-related changes in the kidney (hyaline droplets; 60 and 300 ppm) and the spleen (erythroid cell hyperplasia and pigment deposition; 300 ppm) of both sexes. Cytoplasmic hyaline droplets in the kidneys were characterized by immunohistochemistry as alpha-2μ-globulin. We propose a chronic, oral no-observable-adverse-effect level (NOAEL) of 2.68 mg/kg BW/day for TNB in the rat, based on the hematological and renal changes.

Five structurally related nitrobenzenes (1,2-dinitrobenzene, 1,3-dinitrobenzene, 1,4-dinitrobenzene, 1,3,5-trinitrobenzene, and picric acid) and Meisenheimer complex [1-(S-glutathionyl)-2,4,6-trinitrocyclohexadienate] were evaluated as possible inhibitors of affinity purified mammalian glutathione transferases (GSTs) isolated from human liver or human term placenta and rat fiver. The results suggest that the degree of GST inhibition depends upon both the chemical in question and the enzyme source. Among the nitrobenzenes tested, 1,3,5-trinitrobenzene (TNB) was found to be the most potent inhibitor of GST activity toward 1-chloro-2,4-dinitrobenzene (CDNB) from all the sources, whereas 1,3-dinitrobenzene (1,3-DNB) was the least effective. TNB-caused inhibition of GST activity toward CDNB appeared to be isozyme specific in that compared to the enzyme from human term placenta (GSTP1–1), the degree of inhibition of the mixture of GST isozymes present in the fivers of adult rats and humans was low. The enzyme assays conducted with 3,4-dichloro-1-nitrobenzene (DCNB), ethacrynic acid (EA), and 4-nitropyridinei N-oxide also suggested the isozymespecific inhibition of rat fiver GST activity by TNB. The nature of TNB-caused inhibition of GSTP1–1 was competitive with respect to CDNB and yielded a Ki value of 12.5 θ M. With EA, a specific substrate for GSTP1–1, an IC50value of ∼ 16 θ M was estimated for the GSTP1–1 inhibition by TNB. The Meisenheimer complex, the product of nonenzymatic GSH conjugation with TNB by different GSTs, was found to be the most potent inhibitor of mammalian GSTs, and IC50values ranged between 1 and 4 θ M when the enzyme activity was assayedusing CDNB. The nature of GSTP1–1 inhibition was noncompetitive with respect to CDNB, with a Ki value of 1 θ M for Meisenheimer complex. Although a precise mechanism was not identified, it is postulated that GSH depletion and/or GST inhibition may contribute, at least partly, to the target organ toxicity caused by exposures of animals to different nitrobenzenes reported in the literature.

The contamination of soil and water with munitions chemicals and their degradation products has been reported at certain munitions production waste disposal sites and at certain Army installations. The effects of 2,4,6-trinitrotoluene (TNT) on wild cotton rats ( Sigmodon hispidus) were evaluated to identify target organ toxicity that could be used to develop biomarkers for exposure assessment for ecological and health risks. The oral LD50 values for TNT in corn oil were 607 and 767 mg/kg body weights for male and female cotton rats respectively. Hematological, pathological, and biochemical effects of TNT were determined after daily oral gavage of TNT in corn oil at doses of 0, 75.9, 151.8, and 303.5 (males) or 0, 96, 192, and 384 mg/kg (females) for 7 days. Cotton rats treated with TNT showed an increase in spleen weights in males (303.5 mg/kg) and in females (192 and 384 mg/kg). Cotton rats of both sexes treated for 7 days with TNT had marked hemolytic anemia with reduced erythrocytes, hemoglobin, and hematocrit in high-dose groups; methemoglobin levels were elevated significantly in males at mid and high dose. Hepatic drug metabolizing enzyme analysis revealed that microsomal O-dealkylation of methoxy, ethoxy, and pentoxy resorufin were elevated in male (high dose) cotton rats. The activity of hepatic glutathione S-transferases (GST) was significantly elevated in male (mid and high dose) and female (all doses) cotton rats exposed to TNT. Histopathological analysis of spleen revealed mild to marked splenic congestion with mild extramedullary hematopoiesis, hemosiderosis, and lymphoid hyperplasia in male and female cotton rats treated with TNT (all doses). Liver weights were increased in males (mid and high dose) and in females (high-dose group). In the high-dose groups, histological changes in liver (mild to moderate hepatocellular hypertrophy, increased hemosiderin pigment in Kupffer cells) in both sexes, and in testis (premature exfoliation of spermatozoa from dilated seminiferous tubules) were observed (mid and high dose). These results suggest that hepatic GST and hemolytic anemia may be biomarkers in cotton rats of terrestrial contamination with TNT or other nitroaromatic explosive compounds.

1,3-Dinitrobenzene (DNB) and 1,3,5-trinitrobenzene (TNB) are used primarily in explosive compositions and munitions and have been detected as environmental contaminants of surface waters as well as ground waters near production waste disposal sites. Hemoglobin (Hb) adducts have recently been proposed as biological markers of exposure assessment for various environmental compounds, including nitroaromatics. In the present study, we have investigated the formation of DNB and TNB hemoglobin adducts in vivo and in vitro in the blood of shrew (Cryptotis parva). DNB and TNB hemoglobin adducts were detected by gas chromatography/mass spectrometry (GC/MS) after either basic (0.1 N NaOH) or acid (2 N HCl) hydrolysis followed by organic solvent extraction and derivatization of the corresponding amines. The levels of DNB-Hb adducts detected after basic hydrolysis (238.7 & pm; 50.2 pg/mg Hb) are higher than the corresponding levels detected after acid hydrolysis (52.5 & pm; 16.2 pg/mg Hb). For the TNB-Hb the levels after acid hydrolysis (132.2 & pm; 37.8 pg/mg Hb) are higher than the levels detected after basic hydrolysis (44.7 & pm; 15.3 pg-mg Hb). These results demonstrate the effectiveness of the hemoglobin adduct model for monitoring exposure to nitroaromatics.

The toxicity of tetryl (N-methyl-N,2,4,6–tetranitroaniline) in male and female F344 rats was evaluated after adminstration in the diet for 14 or 90 days. The 14–day study diet concentrations used were 0, 500, 1250, 2000, 2500, and 5000 ppm; the 90–day study diet concentrations were 0, 200, 1000, and 3000 ppm tetryl in the diet. The calculated average daily tetryl intake was 32.1, 82.5, 130.3, 178.9, and 374.4 mg/kg body weight (BW) for females and 31.8, 80.0, 121.0, 170.5, and 349.7 mg/kg BW for males in the 14–day study. For the 90–day studies, the daily tetryl intake was 14.2, 68.8, and 199.0 mg/kg BW for females and 13.0, 62.4, and 179.6 mg/kg BW for males. In the 14–day study, there was a significant decrease in body weights (males), whereas relative(organ/body weight) liver and spleen (females), and kidney (males) weights were significantly increased in the 5000–ppm dose group. Hematological effects observed were decreased hemoglobin and hematocrit and an increased number of reticulocyts in females (2000 to 5000 ppm). Methemoglobin levels in males (2000 to 5000 ppm) and females (5000 ppm) and total blood protein and albumin levels in all groups of males and females (except 500 ppm) were significantly increased. Histopathological changes were observed in kidneys (deposition of cytoplasmic droplets) of all dose groups of male rats. In the sub–chronic (90–day) study, feed intake was reduced in all dose groups, but a significant decrease in terminal body weights was observed in females (1000 and 3000 ppm) and males (3000 ppm). An increase in the relative liver, kidney (1000–3000 ppm), and spleen (3000 ppm) weights were noted in both sexes. The hemoglobin content and red blood cell count were decreased whereas the reticulocyte count was elevated (3000 ppm) in both sexes at 45 and 90 days. Methemoglobin levels were increased in both sexes (1000 and 3000 ppm). Histopathologicalchanges were noted in the spleen (pigment deposition and erythroid cell hyperplasia) of both sexes (3000 ppm) and kidneys (tubular degeneration and cytoplasmic droplets containing alpha-2-micro globulin) of male rats (1000 to 3000 ppm). A no observed adverse effect level (NOAEL) for both sexes was 13 mg/kg BW/day was determined.

The percutaneous absorption of 1,3,5-trinitrobenzene (TNB) was studied in viable skin from hairless guinea pigs (HGP), Fischer 344 rats and humans. Skin was dermatomed and assembled in flow-through diffusion cells followed by TNB application in either an acetone or a water vehicle. Skin absorption was expressed as the percentage of applied dose absorbed into skin and receptor fluid within 24 h. Rapid absorption of TNB by rodent skin was obtained with both vehicles. For HGP skin, TNB absorption was 72.7+/-5.5% in the acetone vehicle and 82.3+/-4.5% in the water vehicle. For rat skin, TNB absorption was 61.0+/-4.1% (acetone) and 66.5+/-4.1% (water). Absorption of TNB from acetone was significantly reduced (38.0+/-11.0%, P = 0.0118) in human skin, but absorption from water remained high (75.5+/-10.8%). Little TNB remained in skin when a thin (200 microm) dermatome section was used (HGP and human skin). A thicker dermatome section was required (350 microm) with haired rat skin, and 13-21% of the absorbed radioactivity remained in the skin at 24 h. Rodent skin did not simulate satisfactorily the barrier properties of human skin when TNB absorption was reduced by application in a volatile solvent.

The subchronic toxicity of 1,3,5-trinitrobenzene (TNB) in male and female Fischer 344 rats was evaluated by feeding a powdered certified laboratory diet containing 0, 66.7, 400 and 800 mg TNBl kg diet for 90 days. The calculated average TNB intake was 4.29, 24.70, and 49.28 mg/kg body weight (BW)day for females and 3.91, 22.73, and 44.16 mg/ kg BWI day for males. Food intake in the 400 and 800 mg/kg diet dose groups of both sexes was decreased throughout the study and resulted in a significant decrease in absolute body weights. A significant decrease in relative testicular weights and a significant increase in the relative liver weight were observed in male rats receiving 400 or 800 mg TNB/kg diet. A significant increase in the relative spleen weights of both sexes receiving 400 or 800 mg TNB diet was noted. The relative liver weight was also increased only in female rats maintained on the 800 mg TNB diet. Histopathological examinations revealed that the susceptible organs for TNB toxicity were kidney (hyaline droplets) in all male dose groups and testes (seminiferous tubular degeneration) in rats receiving 400 and 800 mg TNB diet groups. The spleen was also affected (extramedullary hematopoiesis) in both sexes in the 400 and 800 mg dose groups. Hematological studies at both 45 (data not given) and 90 days in the 400 and 800 mg dose groups indicated decreased values for red blood cell counts and hemoglobin content, while reticulocytes and methemoglobin levels were increased. Clinical chemistry parameters were unaffected. Based on kidney toxicity and hematological effects, a Low Observed Adverse Effect Level (LOAEL) of 3.91 mg/kg BWI day was suggested for subchronic toxicity studies on TNB.

The remedial investigation/feasibility studies conducted at certain Army installations showed a need to clean up contaminated sites, where high levels of ammunition chemicals such as 2,4,6-trinitrotoluene (TNT), 1,3,5-trinitrobenzene (TNB), 1,3-dinitrobenzene (DNB), and their degradation products/metabolites were detected in surface soil and groundwater. TNB is a photodegradation product of TNT; it is not easily degraded, and persists in the environment. The toxicity data on TNB are scanty. Hence the U.S. Environmental Protection Agency in 1988 (U.S. EPA, 1997) developed a reference dose (RfD) for TNB (0.00005 mg/kg/d for chronic toxicity) based on the toxicity of DNB, which is structurally similar to TNB. Since then we have completed acute, subacute, sub-chronic, chronic, reproductive, and developmental toxicity studies and toxicokinetics studies. We have reviewed the mammalian toxicity data for TNB and have determined the no observed adverse effect levels (NOAEL) and low observed adverse effect levels (LOAEL) for subchronic, chronic, reproductive, and developmental toxicity. Based on the newly determined NOAEL and LOAEL values, we have now developed a new RfD for TNB (0.03 mg/kg/d), based on the chronic toxic effects on hematology and histopatho-logical changes in testes and kidney.

The applicability of PCNA as a tool for the analysis of germ cells in rats treated with 1,3,5-trinitrobenzene (TNB), a potent testicular toxicant, was evaluated. Male Fischer 344 (F344) rats were gavaged with TNB at 71 mg/kg or with corn oil (vehicle). Rats were killed after 10 daily oral doses or were allowed to recover for 10 or 30 d after the 10 doses. Testes from control rats, treated rats, and rats allowed to recover were immunohistochemically stained for PCNA. PCNA labeling in the control rats was confined to the nuclei of spermatogonia, pachytene spermatocytes, and nuclei of elongate spermatocytes. Conventional (hematoxylin and eosin) staining of testes from rats treated with TNB at 71 mg/kg for 10 d revealed loss of germ cells and cessation of spermatogenesis. Immunohistochemical staining of sections from these treated rats revealed only PCNA-positive spermatogonia. Rats allowed a 10-d recovery had both spermatogonial and spermatocytic staining, indicating partial restoration of germ-cell population. In rats allowed to recover for 30 d, the PCNA staining pattern was identical to the control rats. These results indicate that PCNA can be used to assess the proliferative status of spermatogonia (germ cells) in rodent testes exposed to testicular toxicants.

Male and female Fischer-344 (F-344) and male NCI-Black-Reiter (NBR) rats were dosed with 0, 35.5, or 71 mg 1,3,5-trinitrobenzene (TNB)/kg/day for 10 days. Male F-344 rats were dosed with TNB (0 and 35.5 mg/kg) for 20 and 30 days. Hematoxylin and eosin and Mallory-Heidenhain stains and alpha-2u-globulin and proliferating cell nuclear antigen immunohistochemical stains were performed on kidney sections. All treated male F-344 rats exhibited dose-related accumulation of hyaline droplets containing alpha-2u-globulin in proximal tubules. The kidney weights were significantly increased in male and female rats treated with TNB. Significant increases in cell proliferation in proximal tubules were observed in male F-344 rats. Renal changes observed in TNB-treated rats appeared identical to those from other chemicals that induce alpha-2u-globulin nephropathy in male rats. No hyaline droplet accumulation was found in female F-344 and male NBR rats at any doses. We can conclude that TNB induces dose-related exacerbation of hyaline droplets containing alpha-2u-globulin in male rat kidney and subsequent cell proliferation.

We studied in vitro dermal penetration of the newly developed munition, IMX-101 (Insensitive Munitions eXplosive) as whole composition and its individual components; 2-4-dinitroanisole (DNAN), 3-nitro-1,2,4,-triazole-5-one (NTO), and nitroguanidine (NQ) in static Franz diffusion cells. The penetration rate was calculated (micrograms/sq cm/hr) for each chemical. The analysis of absorbed chemical in the receptor fluid data showed that steady state of fluxes infinite dose (100 mg) of neat NTO, DNAN, and NQ were 338.2, 1.1, and 31.3 micrograms/sq cm/hr, respectively. The IMX-101 powder (100 mg) had a rate of flux for NTO, DNAN, and NQ were 135.9, 1.18, and 236.3 micrograms/sq cm/hr, respectively. The penetration of IMX-101 (10 mg) with Tyvek showed the penetration flux of NTO, DNAN, and NQ were 102.5, 1.9, and 46.4 micrograms/sq cm/hr, respectively. These estimated values in vitro human model will be used to evaluate health risk associated with dermal exposure.

Toxic effects of 1,3,5-trinitrobenzene (TNB) in male and female rats were evaluated by feeding powdered certified laboratory chow diet supplemented with varied concentrations of TNB (0, 50, 200, 400, 800 and 1200 mg kg -1 diet) for 14 days. Food intake by female rats in 400, 800 and 1200 mg TNB diet groups was reduced and resulted in a significant decrease in absolute body weights (BW). Food and water consumption by male rats in high-dose groups (800 and 1200 mg TNB kg -1 diet) was also reduced and resulted in a significant decrease in body weight. The calculated average TNB intake (from 1200 mg TNB kg -1 diet) was 92mg kg -1 BW day-' for male rats and 80mg kg -1 BW day-' for females. A decrease in testicular weight in males and an increase in spleen weight of both sexes in high-dose groups was noted. In addition, histopathological examinations revealed that the susceptible organs for TNB toxicity were kidney (hyaline droplets), spleen (extramedullary hematopoiesis), brain (hemorrhage, malacia and gliosis) and testes (seminiferous tubular degeneration). Hematology and clinical chemistry studies indicated a decrease in red blood cell count and hematocrit, a decrease in alkaline phosphatase, an increase in Heinz bodies and increased methemoglobin concentration as compared to controls in both sexes. A lowest observed adverse effect level of 4.41 mg TNB kg -1 BW day -1 was established based on the findings of this study.

Administration of 1,3,5-trinitrobenzene (TNB) to male Fischer-344 rats produced ataxia after 6 or 7 oral doses (71 mg/kg). Light microscopic examination after 10 days revealed petechial hemorrhages in the brain stem and cerebellum and bilaterally symmetric degeneration and necrosis (malacia) with reactive gliosis in the cerebellar peduncles. The malacia was dorsal and lateral to the fourth ventricle involving the cerebellar nuclei, medial and lateral vestibular nuclei, and inferior colliculi. Blood vessels associated with the lesion had widened Virchow-Robin spaces, occasionally with extravasated erythrocytes. Rats administered daily oral doses of 35.5 mg/kg of TNB for 10 days and 35.5 and 71 mg/kg of TNB for 1 or 4 days did not have brain lesions.

RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) is an explosive used in military applications. It has been detected in ground water surrounding US military installations and at manufacturing facilities. RDX has been shown to produce hepatotoxicity, testicular, and neurological effects in animals, the latter also in humans. The current chronic oral reference dose (RfD) of 0.003 mg/kg/day was derived based on prostate effects in rats. Here, we provide a reevaluation of the risk associated with RDX exposure by examining old and new data and using physiologically based pharmacokinetic (PBPK) modeling approaches. Candidate non-cancer endpoints in rodents were evaluated and the most plausible mode(s) of action were determined. A PBPK model was used to derive appropriate internal doses based on the mode of action, and then a benchmark dose (BMD) and the lower confidence limit on the BMD (BMDL) were determined using these internal doses in animals. Uncertainty factors (UF) were applied to the animal BMDL or no-observed effect level and a human PBPK model was used to determine a human equivalent dose resulting in the candidate RfDs (cRfDs). A proposed chronic RfD of 0.07 mg/kg/day, based on multiple effects observed in rats, was selected from among the cRfDs.

A reversed-phase high-performance liquid chromatographic method for the separation and quantitation of a mixture consisting of nitrobenzene, dinitrobenzene isomers, 1,3,5-trinitrobenzene and their reduction products: aniline, nitroanilines and phenylenediamines has been developed. The method is sensitive and highly reproducible. The mixture is resolved on a Zorbax C 8 column with 0.1% triethylamine and methanol as the mobile phase. The detection limits for individual chemicals at 254 nm are in the range of 25–50 ng.

3-Nitro-1,2,4-triazol-5-one (NTO) is an energetic explosive proposed for use in weapon systems, to reduce the sensitivity of warheads. In order to develop toxicity data for safety assessment, we investigated the genotoxicity of NTO, using a battery of genotoxicity tests, which included the Ames test, Chinese Hamster Ovary (CHO) cell chromosome aberration test, L5178Y TK(+/-) mouse lymphoma mutagenesis test and rat micronucleus test. NTO was not mutagenic in the Ames test or in Escherichia coli (WP2uvrA). NTO did not induce chromosomal aberrations in CHO cells, with or without metabolic activation. In the L5178Y TK(+/-) mouse lymphoma mutagenesis test, all of the NTO-treated cultures had mutant frequencies that were similar to the average frequencies of solvent control-treated cultures, indicating a negative result. Confirmatory tests for the three in vitro tests also produced negative results. The potential in vivo clastogenicity and aneugenicity of NTO was evaluated using the rat peripheral blood micronucleus test. NTO was administered by oral gavage to male and female Sprague-Dawley rats for 14 days at doses up to 2g/kg/day. Flow cytometric analysis of peripheral blood demonstrated no significant induction of micronucleated reticulocytes relative to the vehicle control (PEG-200). These studies reveal that NTO was not genotoxic in either in vitro or in vivo tests and suggest a low risk of genetic hazards associated with exposure.

Cyclotrimethylenetrinitramine (RDX), a compound used widely in bursting-type munitions, is a concern for the U.S. Department of Defense because it has been detected in soil and groundwater at military installations. Dermal absorption of (14)C-RDX from acetone solutions and from two different soils was studied using excised human skin (from surgery) in flow-through diffusion cells. RDX in acetone (10 muL) or in soils (10 mg) was applied to the epidermal surface of the skin (0.64 cm(2)) and allowed to transverse the skin and become dissolved in a reservoir of receptor fluid that was maintained in contact with the dermal surface. The reservoir was of the flow-through type and receptor fluid was pumped at a rate of 1.5 mL/h. Receptor fluid was collected every 6 h for 24 h. Because the bioavailability of a chemical from soils depends on soil composition, dermal absorption of (14)C-RDX from both a low-carbon (1.9%) and a high-carbon (9.5%) soil was assessed. At the conclusion of the experiment, the RDX remaining on the skin was washed with soap and water using cotton swabs, and the radioactivity present in washings was determined. The stratum corneum was removed from the deeper epidermis and radioactivity found in that layer was not considered in calculations of dermal absorption. The dermal absorption of RDX was relatively low. Only about 5.7 +/- 1.9% of the RDX that had been applied in acetone was found in the skin (epidermis and dermis) (3.2 +/- 1.9) and receptor fluid (2.5 +/- 1.8) combined (over the full 24-h duration of the study). The levels of RDX found in the skin layers were stratum corneum 2.1%, epidermis 0.83%, and dermis 0.45%. The total recovery of applied dose (receptor fluid, skin, and washings) was about 80%. The extent of RDX absorption from soil was even lower than from acetone. Approximately 2.6 +/- 1.1% of the RDX applied in the low-carbon soil and 1.4 +/- 0.41% applied in the high-carbon soil was found in receptor fluid and skin in 24 h. The total recovery of the applied dose (receptor fluid, skin, and washings) was about 87% for the low-carbon soil and 94% for the high-carbon soil. Thus, the dermal absorption of RDX from soils was reduced considerably when compared with absorption from acetone and absorption was lower in the high-carbon soil than in the low-carbon soil.

A physiologically based pharmacokinetic (PBPK) model for simulating the kinetics of cyclotrimethylene trinitramine (RDX) in male rats was developed. The model consisted of five compartments interconnected by systemic circulation. The tissue uptake of RDX was described as a perfusion-limited process whereas hepatic clearance and gastrointestinal absorption were described as first-order processes. The physiological parameters for the rat were obtained from the literature whereas the tissue : blood partition coefficients were estimated on the basis of the tissue and blood composition as well as the lipophilicity characteristics of RDX (logP = 0.87). The tissue : blood partition coefficients (brain, 1.4; muscle, 1; fat, 7.55; liver, 1.2) obtained with this algorithmic approach were used without any adjustment, since a focused in vitro study indicated that the relative concentration of RDX in whole blood and plasma is about 1 : 1. An initial estimate of metabolic clearance of RDX (2.2 h(-1) kg(-1)) was obtained by fitting PBPK model simulations to the data on plasma kinetics in rats administered 5.5 mg kg(-1) i.v. The rat PBPK model without any further change in parameter values adequately simulated the blood kinetic data for RDX at much lower doses (0.77 and 1.04 mg (-1) i.v.), collected in this study. The same model, with the incorporation of a first order oral absorption rate constant (K(a) 0.75 h(-1)), reproduced the blood kinetics of RDX in rats receiving a single gavage dose of 1.53 or 2.02 mg kg(-1). Additionally, the model simulated the plasma and blood kinetics of orally administered RDX at a higher dose (100 mg kg(-1)) or lower doses (0.2 or 1.24 mg kg(-1)) in male rats. Overall, the rat PBPK model for RDX with its parameters adequately simulates the blood and plasma kinetic data, obtained following i.v. doses ranging from 0.77 to 5.5 mg kg(-1) as well as oral doses ranging from 0.2 to 100 mg kg(-1).

This project is the first to investigate RDX metabolism in human liver. RDX metabolism was screened in human liver tissues including S9 preparations, microsomes, hepatocytes and several recombinant CYP450 isoforms under aerobic, anaerobic and oxygen reduced conditions. RDX metabolism was also conducted in several animal liver microsomes to compare with human liver microsomes loss of the parent compound (RDX) was determined at 30 and 180 minutes of the incubation and ranked as human (46.6% & 51.8%) rat (40.1% & 47.2%) monkey (34.6% & 35.7%) Pig (25.5% & 33.7%) rabbit (11.6% & 18.0). The data is used to establish Physiologically-based pharmacokinetic (PBPK) models for human useful in risk assessment. Further characterization of the profiles of RDX in vitro metabolism in human and animal liver tissues is proposed. This study developed an in vitro metabolic model which mimics in vivo physiological condition and will be useful in the evaluation of human metabolic fate for novel energetics such as replacement formulations for RDX and for other toxic TICs/TIMs.

Cyclotrimethylenetrinitramine (RDX), a commonly used military explosive, was detected as a contaminant of soil and water at Army facilities and ranges. This study was conducted to determine the relative oral bioavailability of RDX in contaminated soil and to develop a method to derive bioavailability adjustments for risk assessments using rodents. Adult male Sprague-Dawley rats preimplanted with femoral artery catheters were dosed orally with gelatin capsules containing either pure RDX or an equivalent amount of RDX in contaminated soils from Louisiana Army Ammunition Plant (LAAP) (2300 μg/g of soil) or Fort Meade (FM) (670 μg/g of soil). After dosing rats, blood samples were collected from catheters at 2-h intervals (2, 4, 6, 8, 10, and 12) and at 24 and 48 h. RDX levels in the blood were determined by gas chromatography. The results show that the peak absorption of RDX in blood was 6 h for neat RDX (1.24 mg/kg) and for RDX from contaminated soil (1.24 mg/kg) of LAAP. Rats dosed with RDX-contaminated FM soil (0.2 mg/kg) showed peak levels of RDX in blood at 6 h, whereas their counterparts that received an identical dose (0.2 mg/kg) of neat RDX showed peak absorption at 4 h. The blood levels of absorbed RDX from LAAP soil were about 25% less than for neat RDX, whereas the bioavailability of RDX from FM soils was about 15% less than that seen in rats treated with neat RDX (0.2 mg/kg). The oral bioavailability in rats fed RDX in LAAP soil and the FM soil was reduced with the neat compound but decrease in bioavailability varied with the soil type.

Thiodiglycol (TG), a hydrolysis product of sulfur mustard (HD), is a potential contaminant of soil and water at certain military sites. To establish developmental toxicity criteria for TG, an oral developmental toxicity study was conducted in Sprague-Dawley rats. Neat thiodiglycol (99.9 %) was administered orally to mated female rats from gestation days (GDs) 5 through 19. The day of positive mating was considered day 0. A pilot study was conducted with TG at dose levels 250, 500, 1000, 2000, or 5000 mg/kg to select suitable doses for the main study. In the main study, three groups of rats (25/group) received TG by gavage at dose levels of 430, 1290, or 3870 mg/kg/day. A fourth group served as a sham control. On day 20 of gestation, all females were euthanized and a cesarean section performed. Litters were examined for soft tissue and skeletal alterations. Maternal toxicity was limited to dams receiving TG at 3870 mg/kg/day. At this dose, body weights and food consumption were reduced during certain periods of gestation. Fetuses derived from those dams exhibited a nonstatistically significant increased incidence of variations when compared to controls. Fetal body weights in the 3870 mg/kg/day group were significantly lower than controls. There was no increased incidence of anomalies when thiodiglycol-treated fetuses were compared to controls. It was concluded that TG did not produce terata. Developmental toxicity (decreased fetal weights and associated delays in development) occurred only at the maternally toxic dose of 3870 mg/kg. It appears that 1290 mg/kg/day could be considered no observed adverse effect level (NOAEL) for oral developmental toxicity. The lowest observed adverse effect level (LOAEL) was 3870 mg/kg for maternal toxicity.

Hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) has been widely used as an explosive in U.S. army munitions formulations since World War II. Two-year carcinogenicity studies revealed RDX to be noncarcinogenic in two strains of rats, but a 2-year carcinogenicity study in B6C3F1 mice revealed an increased incidence of hepatocellular neoplasms in females. Based on results of the study in B6C3F1 mice, RDX has been classified as a possible carcinogen. The authors reevaluated the archived histological sections from the B6C3F1 mouse study, using current histopathologic diagnostic criteria and interpretations. The earlier evaluation showed a statistically significant increase in the incidence of hepatocellular adenoma/carcinoma in female mice from the three highest dose groups (7, 35, and 175/100 mg/kg/day). The revaluation yielded a slightly lower incidence at each of the dose levels in female mice. The reduced number of hepatocellular neoplasms was largely due to reclassification of hepatocellular adenomas as foci of cytoplasmic alteration, in compliance with current diagnostic criteria. The reevaluation was reviewed by a pathology working group (PWG), which arrived at a consensus classification of each lesion. Based on the consensus diagnoses of the PWG, only one female group (35 mg/kg/day) showed a significant increase when compared to controls. The incidence of hepatocellular neoplasms for all groups, including the 35 mg/kg/day group, was within the reported incidence range for spontaneous hepatocellular neoplasms in female B6C3F1 mice. The increased incidence of hepatocellular neoplasms in female mice given RDX at 35 mg/kg/day was interpreted as equivocal evidence of a carcinogenic effect.

Hexahydro-1,3,5-trinitro-1,3,5-triazine, a polynitramine compound, commonly known as RDX, has been used as an explosive in military munitions formulations since World War II. There is considerable data available regarding the toxicity and carcinogenicity of RDX. It has been classified as a possible carcinogen (U.S. Environmental Protection Agency, Integrated Risk Information System, 2005, www.epa.gov/IRIS/subst/0313.htm ). In order to better understand its gentoxic potential, the authors conducted the in vitro mouse lymphoma forward mutation and the in vivo mouse bone marrow micronucleus assays. Pure RDX (99.99%) at concentrations ranging from 3.93 to 500 μg/ml showed no cytotoxicity and no mutagenicity in forward mutations at the thymidine kinase (TK) locus in L5178Y mouse lymphoma cells, with and without metabolic activation. This finding was also confirmed by repeat assays under identical conditions. In addition, RDX did not induce micronuclei in mouse bone marrow cells when tested to the maximum tolerated dose of 250 mg/kg in male mice. These results show that RDX was not mutagenic in these in vitro and in vivo mammalian systems.

Sulfur mustard (HD) undergoes hydrolysis to form various products such as thiodiglycol (TG) in biological and environmental systems. TG is a precursor in the production of HD and it is also considered as a “Schedule 2” compound (dual-use chemicals with low to moderate commercial use and high-risk precursors). Several toxicological studies on TG were conducted to assess environmental and health effects. The oral LD50 values were >5000 mg/kg in rats. It was a mild skin and moderate ocular irritant and was not a skin sensitizer in animals. It was not mutagenic in Ames Salmonella, Escherichia coli, mouse lymphoma, and in vivo mouse micronucleus assays, but it induced chromosomal aberrations in Chinese hamster ovarian (CHO) cells. A 90-day oral subchronic toxicity study with neat TG at doses of 0, 50, 500, and 5000 mg/kg/day (5 days/week) in Sprague-Dawley rats results show that there are no treatment-related changes in food consumption, hematology, and clinical chemistry in rats of either sex. The body weights of both sexes were significantly lower than controls at 5000 mg/kg/day. Significant changes were also noted in both sexes in absolute weights of kidneys, kidney to body weight ratios, and kidney to brain weight ratios, in the high-dose group. The no-observed-adverse-effect level (NOAEL) for oral toxicity was 500 mg/kg/day. The developmental toxicity conducted at 0, 430, 1290, and 3870 mg/kg by oral gavage showed maternal toxicity in dams receiving 3870 mg/kg. TG was not a developmental toxicant. The NOAEL for the developmental toxicity in rats was 1290 mg/kg. The provisional oral reference dose (RfD) of 0.4 mg/kg/day was calculated for health risk assessments. The fate of TG in the environment and soil showed biological formation of thiodiglycalic acid with formation of an intermediate ((2-hydroxyethyl)thio)acetic acid. It was slowly biodegraded under anaerobic conditions. It was not toxic to bluegill sunfish at 1000 mg/L and its metabolism and environmental and biochemical effects are summarized.